Most of what is known about the development of the sympathetic nervous system (SNS) is limited to molecules with essential functions such as Nerve Growth Factor (NGF). Since loss of NGF and immediate downstream molecules such as its cognate receptor, TrkA, result in near complete loss of the SNS, they offer little clues to the underlying causes of SNS diseases which are far less severe. Instead studies of genes further downstream of NGF signaling such as Egr3, a transcription factor shown to be regulated by NGF, will likely offer better insight into disease. Deletion of Egr3 in the germline results in mice with abnormalities in SNS development and physiologic dysautonomia bearing similarities to human diseases. The proposed study will further extend our understanding of Egr3 function by (1) testing the hypothesis that Egr3 is a sympathetic neuron autonomous regulator of SNS development and (2) defining the role it has independent of NGF signaling in sympathetic neurons. This will be accomplished by generating novel mouse models that will specifically delete Egr3 in sympathetic neurons and specifically over-express Egr3 in sympathetic neurons. Morphological and physiological assays will then be used to determine whether cell-autonomous manipulation of Egr3 affects SNS development. Along with determining the necessity of Egr3 in SNS development, studies will be done to better understand the mechanisms by which it affects sympathetic neuron differentiation. Thus, a newly identified Egr3 target gene, hs3st2, which is involved in heparan sulfate proteoglycan (HSPG) synthesis, will be further examined. Since our initial studies have confirmed that Egr3 can regulate hs3st2 and previous studies have implicated the importance of HSPGs in nervous system development, the second aim of this proposed study will be to (1) better characterize the regulation of hs3st2 by Egr3 and (2) determine whether hs3st2 has an important role in SNS development. Various in vitro assays will be used to determine whether regulation of hs3st2 by Egr3 is either direct or indirect and if it is direct, the exact location of binding will be established. To determine whether hs3st2 is required for SNS development, hs3st2 germline null mice will be examined to assess whether they have any SNS abnormalities that are reminiscent of those seen in Egr3-deficient mice. PUBLIC HEALTH RELEVANCE: The SNS is susceptible to a variety of developmental and degenerative diseases that afflict millions of people worldwide. A few of the SNS diseases have been reliably associated with specific gene mutations, but the vast majority of them have no identifiable cause, thus highlighting our still limited knowledge of the details of SNS development. Egr3 is a gene shown to be involved in SNS development and thus by further elucidating the mechanisms through which it acts we will gain a better understanding of how the SNS develops and how its function can be impaired by disease.